CN101003800A - Cloning using donor nuclei from non-serum starved, differentiated cells - Google Patents
Cloning using donor nuclei from non-serum starved, differentiated cells Download PDFInfo
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- CN101003800A CN101003800A CNA2007100081920A CN200710008192A CN101003800A CN 101003800 A CN101003800 A CN 101003800A CN A2007100081920 A CNA2007100081920 A CN A2007100081920A CN 200710008192 A CN200710008192 A CN 200710008192A CN 101003800 A CN101003800 A CN 101003800A
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Abstract
An improved method of nuclear transfer involving the transplantation of donor differentiated cell nuclei from non-serum starved cells into enucleated oocytes of the same species as the donor cell is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring, and for production of isogenic CICM cells, including human isogenic embryonic or stem cells. Production of genetically engineered or transgenic mammalian embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.
Description
It is 98807691.8 that the application of this division is based on application number, and the applying date is on June 24th, 1998, and denomination of invention is divided an application for the Chinese patent application of " use from the donorcells nuclear of non-serum starved noble cells and clone ".
Invention field
The present invention relates to go into from the nuclear transplantation Mammals of the mammalian cell of non-serum starved, differentiation cloning process with donorcells nuclear phase stoning mammal ovocyte of the same race.This nucleus is carried out reprography to instruct clone's fetal development, it can be implanted into female receptor then to produce fetus or offspring or to be used to produce the inner cell mass cell (CICM) of cultivation.This clone's embryo also can combine with the embryo of fertilization to produce chimeric embryo, fetus and/or offspring.
Background of invention
Existing report uses inner cell mass (ICM) cell of ungulate to carry out nuclear transplantation.For example, the mature oocyte that discloses in Mol.Reprod.Dev.38:264-267 (1994) by the microinjection of cracked donorcells being gone into stoning such as Collas carries out the nuclear transplantation of ox ICMs.Collas etc. disclose wherein four-head pregnancy and two are produced.And Keefer etc. are at Biol.Reprod., when disclosing in the nuclear transplantation method blastocyte implantation that the ICM cell with ox produces as donor nuclei among the 50:935-939 (1994) and going into the acceptor of ox, have produced some offsprings that can survive.And Sims etc. report in institute of NAS and disclose among the 90:6143-6147 (1993) by will producing calf at the mature oocyte that the ox ICM of external Short-term Culture nucleus is transferred to stoning.
Can produce the lamb (Campbell etc., nature, 380:64-68 (1996)) that can survive behind the placenta cells nuclear that also has the transplanting of reporting to cultivate.Further, generation (Stice etc., Biol.Reprod, the 54:100-110 (1996) of paotoblastic use of ox multipotency and chimeric fetus in the nuclear transplantation of existing report; Collas etc., Mol.Reprod.Dev., 38:264-267 (1994)).Proof granulosa cells such as Collas (one-tenth somatocyte) can be used for the ox cloning process to produce the embryo.But, fail to prove early stage (blastocyst stage) growth afterwards of embryo.And granulosa cell is difficult for cultivating and only can be from female acquisition.Collas etc. do not attempt breeding granulosa cell or attempt those cells of genetic modification in cultivation.Wilmut etc. (nature, 365:810-813 (1997)) produce nuclear transplantation sheep offspring from fetal fibroblast, and have produced an offspring from adult sheep cell.
In the field that produces transgenic pig, also have problems.By existing method, allogeneic dna sequence DNA is introduced in early embryo or the embryonic lineage that is divided into various cell types in the fetus and finally develops into transgenic animal.But producing transgenic animal needs many early embryos, so this method is unusual poor efficiency.And, the simple and effective means of screening transgenosis embryo before the female gonosome of agency put into embryo by spended time and funds not.In addition, the gene targeting technology is not easy to finish with the early embryo transgenic technology.
Embryonic stem cell in the mouse makes the researchist can screen transgenic cell and carries out gene targeting.This causes comparing and will using genetic engineering more with other transgenic technology.But, embryonic stem cell line and other embryonic lineage must be maintained undifferentiated state, this needs feeder layer and/or add cytokine in substratum.Even taked these preventive measures, spontaneous differentiation still can often take place and can not produce transgenic progeny by existing obtainable method in these cells.And some embryonic lineages must be bred in the mode that is unfavorable for the gene targeting method.
The method of dried (ES) clone of external acquisition embryo is well known from the previous pre-mouse embryo of transplanting.(see Evans etc. for example, nature, 29:154-156 (1981); Martin, institute of NAS newspaper, 78:7634-7638 (1981)).If because fibroblastic feeder layer (Evans etc., Id.) or differentiation inhibition source (Smith etc., biology progress, 121:1-9 (1987)) exist, the ES cell is gone down to posterity with undifferentiated state.
Reported in the past that the ES cell had many application.For example, reported that the ES cell can be used as the external model of differentiation, be particularly useful for studying the gene that relates in the early development adjusting.When mouse ES cells being introduced the mouse embryo of pre-transplanting, can produce germline mosaic, thereby prove their versatility (Bradley etc., nature, 309:255-256 (1984)).
Because the ES cell is delivered to follow-on ability with its genome, they have the potential use that the ES cell that has or do not have a required genetic modification by use carries out the operation of livestock animals kind system.And, livestock animals if any the hoof animal in, come to transplant in advance freely growth (Smith etc., Biol.Reprod., 40:1027-1035 (1989) that endorsing of domestic animal embryo promotes enucleation oocyte; With Keefer etc., Biol.Reprod., 50:935-939 (1994)).On the contrary, it is reported that the nuclear that surpasses the embryo of 8 cell stages from mouse does not promote the growth (Cheong etc., Biol.Reprod., 48:958 (1993)) of enucleation oocyte after transfer.Therefore, be unusual ideal from the ES cell of livestock animals, because they can provide through the potential source of the totipotency donor nuclei of genetic manipulation or be used for the nuclear transplantation method.
Some research groups have reported the separation that is called the versatility embryonic lineage.For example, Notarianni etc. are at J.Reprod.Fert.Suppl., reported the foundation from the stable totipotent cell of thinking of pig and sheep blastocyst system among the 43:255-260 (1991), this clone shows some and similar morphology and growth characteristics of cell from the primary culture of the isolating inner cell mass of sheep blastocyst immunosurgery.Simultaneously, Notarianni etc. are also at J.Reprod.Fert.Suppl., and disclosing among the 41:51-56 (1990) from inferring of pig blastocyst is the keeping and break up of culture of totipotency embryonic lineage.Gerfen etc. are in Animal Biotechnology, and 6 (1): disclose among the 1-14 (1995) from the pig blastocyst and separated embryonic lineage.These cells can be in the inoblast feeder layer of the mouse embryo of working conditions substratum not stable maintenance, and it is reported and in culturing process, be divided into several different cell types.
And Saito etc. are at Roux ' s Arch.Dev.Biol., the ox embryonic stem cell like cell that report is cultivated among the 201:134-141 (1992) three generations of can surviving, but dead the 4th back of going down to posterity.Hahdyside etc. disclose the inner cell mass of cultivating the isolating sheep embryo of immunosurgery under the condition of separation from the mouse ES cells system of mouse ICMs at Roux ' s Arch.Dev.Biol. among the 196:185-190 (1987).Handyside etc. are reported under such condition, and sheep ICMs adheres to, spreads the zone of the concurrent ES of bringing out cell sample and endoderm cell's like cell, but only have the entoderm like cell obviously as seen after the cultivation of carrying out the longer time.
Recently, Cherny etc. are at Theriogenology, have reported among the 41:175 (1994) that the genitaloid clone of versatility ox of calling oneself can keep in the long-term cultivation process.These cells after cultivating about 7 days, produce the positive ES sample colony of alkaline phosphatase (AP) dyeing, and performance forms the ability of embryoid, and spontaneously are divided at least two kinds of different cell types.It is reported that these cells also express the mRNA of transcription factor OCT4, OCT6 and HES1, this be sure of it is specially by the pattern of the homeobox gene of ES cell expressing.
And, recent Campbell etc. are at nature, reported among the 380:64-68 (1996) comfortable in the future promote to cultivate under the isolating condition of ES clone in the mouse 9 day age the sheep embryo placenta (ED) cell of cultivation carry out can producing the lamb of survival after the nuclear transplantation.The author reaches a conclusion, and the ED cell of sheep embryo was totipotent after the process nuclear transplantation from 9 day age, and this totipotency can be kept in culturing process.
Van Stekelenburg-Hamers etc. have reported separation that is called permanent cell line and CHARACTERISTICS IDENTIFICATION from ox blastocyst inner cell mass cell at Mol.Reprod.Dev. among the 40:444-454 (1995).The author under different condition, separate and cultivate from the ICM of the ox blastocyst in 8 or 9 day age with measure any helper and substratum promote ox ICM cell adhere to and grow in the most effective.They reach a conclusion adhering to of the ICM cell cultivated and grow and can be enhanced by the substratum that uses the additional serum (rather than standard serum) with activated carbon-desorb of STO (l cell) helper (the intrauterine chrotoplast that replaces ox) and use.Yet Van Stekelenburg etc. has reported that their clone more is similar to epithelial cell rather than versatility ICM cell.
Smith equals disclosed WO 94/24274 on October 27th, 1994, Evans and equals April 5 nineteen ninety disclosed WO 90/03432 and equal on November 24th, 1994 disclosed WO 94/26889 with Wheeler and reported and think the separating of the stem cell animal that can be used for obtaining transgenic animal, screening and breeding.Evans etc. also reported from pig and ox species think to produce transgenic animal useful be called deriving of multipotent stem cells.And Wheeler equals on November 24th, 1994 disclosed WO 94/26884 and also discloses and think to preparing the chimeric embryonic stem cell useful with genetically modified ungulate.
Therefore, based on foregoing content, can obviously find out many groups because ES clone in clone or the generation of transgenosis embryo and the potential application in the nuclear transplantation, has attempted preparing ES clone.
Although the existing report of former document still needs improving one's methods of cloning mammal.
Purpose of the invention and overview
New and the improved method that the purpose of this invention is to provide the Mammals (as embryo, fetus and offspring) that is used to produce the clone.
The present invention's purpose more specifically provides the novel method of the nuclear transplantation of mammalian cell non-serum starved, differentiation being gone into enucleation oocyte mutually of the same race that relates to that is used for cloning mammal.
Another object of the present invention provides the method that is used to breed the Adult Mammals with certified hereditary superiority or other ideal character.
Another object of the present invention provides and is used to produce improving one's methods of genetic engineering or genetically modified Mammals (being embryo, fetus, offspring).The present invention also provides genetic engineering or the genetically modified Mammals that produces by this method.
Purpose more specifically of the present invention provides by inserting, remove or modify the target DNA sequence in mammalian cell that is breaking up before cell that uses differentiation or the nucleus formation NT unit or nucleus and produces genetic engineering or genetically modified mammiferous method.The present invention also provides genetic engineering or the genetically modified Mammals that produces in this way.
Another object of the present invention provides by the nuclear transplantation of the cell of non-serum starved, genetically modified, differentiation being gone into noble cells enucleated oocyte mutually of the same race and produces genetic engineering or genetically modified mammiferous method.The present invention also provides genetic engineering or the genetically modified Mammals that produces in this way.
Another object of the present invention provides to relate to goes into the novel method that produces Mammals CICM cell with noble cells enucleation oocyte mutually of the same race with the nuclear transplantation of the cell of non-serum starved, differentiation.
Another object of the present invention provides the CICM cell that produces with the cell that breaks up enucleation oocyte mutually of the same race by the nuclear transplantation of the mammalian cell of non-serum starved, differentiation is gone into.
The present invention's purpose more specifically provides and relates to the method that the human oocyte that becomes somatic nuclear transplantation to go into stoning non-serum starved people's cell such as people produces people CICM cell.
Another object of the present invention is to use such CICM cell to be used for the treatment of or diagnoses.
Specific purposes of the present invention be to use such CICM cell comprise the CICM cell of people and ungulate be used for the treatment of or diagnose any wherein cell, tissue or organ transplantation the treatment on or the diagnosis on useful disease.These CICM cells can use in mutually of the same race or cross-fertilize seed.
Another object of the present invention is to use and derives from the NT embryo, fetus or offspring's cell or tissue comprises that people and ungulate tissue are used for the treatment of or diagnose any wherein cell, the tissue or organ transplantation the treatment on or the diagnosis on useful i or I comprise Parkinson's disease, Huntington Chorea, alzheimer's disease, ALS, Spinal injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic diseases, heart disease, the cartilage displacement, burn, vascular disease, urethral disease and be used for immune deficiency, bone marrow transplantation, other treatment of diseases such as cancer.These tissues can use these tissues in mutually of the same race or cross-fertilize seed.
Another specific purposes of the present invention are to use the CICM cell that produces according to the present invention to be used to produce cell, tissue or the organ of differentiation.
The present invention's purpose more specifically is to use the people CICM cell that produces according to the present invention to be used to produce people's cell, tissue or the organ of differentiation.
Another specific purposes of the present invention CICM cell that to be external uses produce according to the present invention for example is used for the research of cytodifferentiation and test objective as being used for drug research.
Another object of the present invention provides improving one's methods of transplantation therapy, comprises the application that waits gene or homogenic cell, tissue or organ that produces from the CICM cell that produces according to the present invention.Such methods of treatment comprises that disease for example and damage comprise other treatment of diseases such as the treatment of Parkinson's disease, Huntington Chorea, alzheimer's disease, ALS, Spinal injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic diseases, heart disease, cartilage displacement, burn, vascular disease, urethral disease and immune deficiency, bone marrow transplantation, cancer.
Another object of the present invention provides by insert, remove or modify genetic engineering or the genetically modified CICM cell that the target DNA sequence produces in mammalian cell that is breaking up before cell that uses this differentiation or the nucleus formation NT unit or nucleus.
Another object of the present invention is to use the transgenosis or the genetic engineering CICM cell that produce according to the present invention to be used for gene therapy, to be particularly useful for treating and/or preventing of above-mentioned disease and damage.
Another object of the present invention is to use the CICM cell that produces according to the present invention or transgenosis or engineered CICM cell as the nucleus donor that is used for nuclear transplantation.
Therefore, on the one hand, the invention provides the method that is used for cloning mammal (as embryo, fetus, offspring).Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus stoning mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(iii) the NT unit with described cultivation is implanted into host mammal so that NT unit develops into fetus.
Preferably, cultivation activatory nuclear moves and grows unit until surpassing the 2-cell development stage.
The cell of these fetuses, tissue and/or organ can be advantageously used in cell, tissue and/or filed of organ transplantation.
The present invention also comprises by inserting, remove or modified the target DNA sequence and clone genetic engineering or genetically modified mammiferous method in the mammalian cell of differentiation or nucleus before mammalian cell that will differentiation or nucleus inserting enucleation oocyte.
The present invention also provides Mammals and those the mammiferous offsprings that obtain according to the method described above.
The present invention is preferably used for cloning ungulate.
On the other hand, the invention provides the method that produces the CICM cell.Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus stoning mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(i) activate the nuclear transplantation unit that obtains; And
(iii) cultivate the cell that obtains from the NT unit of described cultivation to obtain the CICM cell.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The CICM cell can be advantageously used in cell, tissue and filed of organ transplantation.
About above-mentioned and hereinafter more obviously other purposes of the present invention, advantage and characteristics, feature of the present invention can more be expressly understood by detailed description and the additional claim with reference to the following preferred embodiment of the invention.
Detailed Description Of The Invention
The invention provides by nuclear transplantation or consideration convey and move improving one's methods of cloning mammal.Among the application, consideration convey moves or nuclear transplantation or NT are used interchangeably.
According to the present invention, will from the nuclear transplantation of the mammalian cell of non-serum starved, differentiation go into donorcells nuclear phase stoning mammal ovocyte of the same race in.Pair cell nuclear carries out reprography to instruct clone's fetal development, it can be implanted into female receptor then to produce fetus and offspring or to be used to produce the CICM cell.Clone's embryo also can combine with the embryo of fertilization to produce chimeric embryo, fetus and/or offspring.
The method of prior art has been used the protoblast type in cloning process.This comprises the work of (Biol.Reprod., 54:100-110,1996) such as Campbell etc. (nature, 380:61-68,1996) and Stice.In two of these researchs, embryonic lineage is less than 10 days embryo from gestation.In these two researchs, cell is kept on feeder layer to prevent the obvious differentiation of the used donorcells of cloning process.Found that the present invention uses fetus or becomes somatocyte effective.
Do not expected that the clone embryos that has fetus or adult donorcells nuclear can grow senior embryo and the fetal state.The science doctrine once was to have only the body early embryo cell type can instruct this type of growth.Do not expected that a large amount of clone embryos can or become somatocyte to produce from the embryo.Also have, it is unexpected that new transgenic embryos clone can obtain this fact from the transgene clone embryo easily.
One-tenth somatocyte and fetal fibroblast from sheep on purpose are used to produce sheep offspring (Wilmut etc., 1997).Yet in that research, the nucleus donorcells of the serum starvation of emphasizing that is to use stationary phase is important for the success of Wilmut cloning process.For the present invention, do not exist such to serum starvation or immobilized requirement.On the contrary, use mammalian cell non-serum starved, differentiation to realize the clone.And according to the present invention, cloning efficiency is same to have nothing to do with using fetus or adult donorcells, and Wilmut etc. (1997) report is lower with the cloning efficiency of adult donorcells.
Therefore, according to the present invention, Mammals comprises that the superior genotypic breeding of ungulate is possible.This will allow to have the breeding of the adult animal of certified gene superiority or other ideal proterties.For example in many important ungulate kinds, will speed up improvement.By the present invention, the countless fetuses that can gather in the crops in cloning process and utilize can be arranged or become somatocyte.This may produce many identical offsprings at short notice.
The method of inferring Wilmut etc. in addition will cause the generation (seeing MacQuitty, Nature Biotech., 15:294 (1997)) of transgenic animal.But, have no reason to infer for example control oneself transfection the somatic nuclear energy of one-tenth of foreign DNA in the nuclear transplantation process, survive.In this, known by manipulation in vitro change mouse embryonic stem (ES) thus the characteristic of cell obtains the ability that their form survival chimeric embryo.Therefore, before the present invention, the clone of transgenic animal does not obtain prediction.
The present invention also can simplify transgenic method by the cell source of energy clonal propagation is operated.This has got rid of the needs that cell maintained undifferentiated state, therefore, comprises that the genetic modification of random integration and gene targeting is easier to realize.This method is also more effective by the nuclear transplantation and the ability of external modification and selection cell are combined the transgenic embryos technology that becomes than former.According to the present invention, these cells can be when the acellular factor, conditioned medium and/or feeder layer clonal propagation, further simplify and help transgenic method.When in cloning process, using transfectional cell, can produce the transgenosis embryo that can develop into fetus and offspring according to the present invention.And these genetically modified clone's embryos can be used for producing CICM clone or other embryonic lineage.Therefore, the present invention has got rid of for helping gene engineering at the external needs of deriving and keeping undifferentiated cell system.
The present invention also can be used for producing CICM cell, fetus or the offspring who can be used for for example cell, tissue and organ transplantation.By getting fetus from animal or become somatocyte and it is used for cloning process, can develop into organ at them and obtain various kinds of cell, tissue and possible organ from clone's fetus in forming.Cell, tissue and organ also can separate from clone's offspring.This method can be provided for " material " source that many medical science and veterinary science therapeutics comprise cell and gene therapy.If these Transplanted cellss get back to cell from animal in, so just avoided immunological rejection.And, because the various kinds of cell type can be from these clone and separate, the chimerism of other method system such as hematopoiesis can be used to avoid mutually of the same race in and plant between the immunological rejection of animal.
Therefore, on the one hand, the invention provides the method for cloning mammal.Usually, Mammals produces by the nuclear transplantation method that comprises the following steps:
(i) obtain the source of the mammalian cell hungry tool of required serum-free, differentiation as donorcells nuclear;
(ii) from the Mammals acquisition ovocyte mutually of the same race with donorcells nuclear derived cell;
(iii) with described ovocyte stoning;
(iv) for example by merging or injecting required noble cells or nuclear transplantation are gone in the non-nucleus egg mother cell to form NT unit;
(v) activate the NT unit that obtains; And
(vi) the NT unit of described cultivation is moved and grow into host mammal so that NT unit develops into fetus.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The present invention also comprises by the method for inserting, removing in mammalian cell that is breaking up before mammalian cell that will break up or the nucleus insertion enucleation oocyte or nucleus or modification target DNA sequence is cloned genetic engineering or genetically modified animal.
The present invention also provides Mammals and those the mammiferous offsprings who obtains according to the method described above.The present invention is preferably used for cloning ungulate.
The present invention further provides the application of NT fetus and NT and chimeric offspring in cell, tissue and filed of organ transplantation.
On the other hand, the invention provides the method that produces the CICM cell.Method comprises:
(i) under the condition that is suitable for the formation of nuclear transplantation unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus stoning mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(iii) cultivate the cell that obtains from the NT unit of described cultivation to obtain the CICM cell.
Preferably, cultivate activatory nuclear transplantation unit until surpassing the 2-cell development stage.
The CICM cell is advantageously used in cell, tissue and filed of organ transplantation or is used to produce fetus or the offspring comprises transgenosis fetus or offspring.
As used herein, fetus is the unborn immature viviparous animal of taking out from the uterus.Therefore, the fetus period of ox is from becoming pregnant back 35 days up to birth.The fetus period of pig is from becoming pregnant back 30 days up to birth.Mammals is an adult from birth to death.
Preferably, at least 2～400 cell sizes are cultivated by NT unit, 4～128 cells more preferably, and most preferably at least about 50 cell sizes.
Consideration convey moves technology or nuclear transfer technology to be had in the literature in many reference of describing and quoting in background of invention description is also arranged.See especially: Campbell etc., Theriogenology, 43:181 (1995); Collas etc., Mol.Report.Dev., 38:264-267 (1994); Keefer etc., Biol.Reprod., 50:935-939 (1994); Sims etc., institute of NAS newspaper, 90:6143-6147 (1993); WO 94/26884; WO 94/24274 and WO90/03432 here quote them as a reference fully.And U.S. Patent number is 4,944,384 and 5,057, and 420 patent has also been described the method for the nuclear transplantation of ox.
Being meant of differentiation has the characteristics different with on every side structure or primary cell or the cell of function.The mammalian cell of differentiation is that those have passed through the body early embryo stages of cell.More specifically, the cell of differentiation is that those are from the cell that passes through the placenta stage (embryogenetic the 10th day of ox) at least.The cell of differentiation can be from ectoderm, mesoderm or entoderm.
Mammalian cell comprises that people's cell can obtain by well-known method.Can be used for mammalian cell of the present invention and comprise for example cell, inoblast, myocardial cell and other muscle cell etc. of epithelial cell, neurocyte, epidermic cell, keratinocyte, hematopoietic cell, melanocyte, chondrocyte, lymphocyte (B and T lymphocyte), red corpuscle, scavenger cell, monocyte, monokaryon.And the mammalian cell that is used for nuclear transplantation can be from acquisitions such as different organs such as skin, lung, pancreas, liver, stomach, intestines, heart, reproductive organ, bladder, kidney, urethra and other urinary organs.These only are the examples of suitable donorcells.Suitable donorcells, promptly useful in the present invention cell can obtain from any cell of health or organ.This comprises all somatocyte or sexual cell.
Inoblast is required cell type, because they can obtain from fetus and the adult animal that grows in large quantities.Inoblast has differentiation to a certain degree, therefore in the past thinks that they are the relatively poor cell types that are used for cloning process.But importantly be, these cells can external with doubling time fast breed easily and can clonal propagation to be used for the method for gene targeting.The present invention be novel also be because used the cell type of differentiation.The present invention is advantageous, because cell is easy in external breeding, genetic modification and screening.
The cloning process of other report (as Wilmut etc., 1997) depends on the cell of using serum starvation.Yet among the present invention, donorcells is not at the serum starvation state.According to (1997) such as Wilmut, the cell of serum starvation is an immobilized,, withdraws from vegetative period that is.Additive method (chemistry, temperature etc.) also can produce resting cell.Used donorcells is not a resting cell among the present invention.
There are sheep, ox, pig, goat, horse, rabbit, cavy, mouse, hamster, rat, primate etc. in the suitable Mammals source of ovocyte.Ovocyte is preferably from ungulate, most preferably obtain from ox.
The method of separating ovocyte is well-known in this area.Basically, this comprises ovary or reproductive tract separation ovocyte from Mammals such as ox.The source of the bovine oocyte that is easy to obtain is the slaughterhouse material.
Successfully use for making as genetic engineering, nuclear transplantation and clone's technology, general ovocyte is as before the recipient cell of nuclear transplantation, and can be by the spermatid fertilization with must be at maturation in vitro before developing into embryo at them.This process generally need be collected immature (I in early stage) ovocyte as the ox ovary that obtains in the slaughterhouse from Mammalian Ovary, and making it ripe until the ovocyte arrival II stage in mid-term in maturation medium before fertilization or stoning, this generally occurs in drew bovine oocyte after about 18-24 hour.To achieve the object of the present invention, this time period is called " ripening stage ".As used herein, for section computing time, " absorption " is meant and draws immature ovocyte from ovary follicle.
And, be successfully used in the nuclear transfer technology at the ovocyte in external mature II stage in mid-term.Basically, sophisticated II ovocyte in mid-term 35 to 48 hours non-super ovulation or superovulated cow or the collection of heifer Chinese and foreign department after oestrus or behind injection human chorionic gonadotrophin (hCG) or the similar hormone.
The success of existing stage of maturity of reporting the ovocyte in stoning and the nuclear transplantation to the NT method is important.(for example seeing Prather etc., differentiation, 48,1-8,1991).Usually, successful Mammals embryo cloning process is can or to be enough to obtain the nuclear that sperm that " activation " be fertilized with the picture processing is handled introducing because be sure of ovocyte in this stage as the acceptor ovocyte with the ovocyte in II stage in mid-term.In domestic animal, the ovocyte pot-life generally is after absorption about 16-52 hour, preferably about 28-42 hour.
For example, can be as Seshagine etc., biology of reproduction 40,544-606, wash immature ovocyte in the HEPE buffered hamster embryo culture medium (HECM) described in 1989, just be placed on then and contain comprising of 50 microlitres of suitable gonad-stimulating hormone such as during the maturation medium of the tissue culture medium (TCM) (TCM) 199 of 10% foetal calf serum of lutropin (LH) and follicle-stimulating hormone (FSH) and estradiol drips under 39 ℃ of thin layer paraffin or the silicon layer.
From about 10-40 hour, and after one section preferably about 16-18 hour fixed maturation time, with the ovocyte stoning.After stoning, preferred mobile ovocyte and before removing the upright cell of ovum, ovocyte is placed the HECM of the Unidasa that contains 1 milligram every milliliter.This can beat or of short duration rotation is finished by repeating to inhale with the suction pipe of pore very.Then, the polar body of the ovocyte that screening is exposed, and according to the existence of polar body and the ovocyte of the metaphase of definite choosing just is used for nuclear transplantation.Stoning subsequently.
Stoning can be undertaken by currently known methods, as being described in 4,994,384 the patent at U.S. Patent number, here quotes as a reference.For example, can with mid-term II ovocyte place optionally that every milliliter of HECM that contains 7.5 microgram cytochalasin Bs (CB) is used for stoning immediately, perhaps can place suitable medium, for example embryo culture medium such as CRlaa, add 10% oestrus bovine serum, a little stonings in evening then, preferably be no more than 24 hours after, and more preferably 16-18 hour.
Available micropipet is removed polar body and peripheral cell matter is finished stoning with coming microsurgery.Screening and Identification goes out those successful non-nucleus egg mother cells.This screening can be observed ovocyte and carry out then by 33342 Hoechst dyeings with every milliliter 1 microgram among the HECM in 10 seconds under uviolizing.Successful then non-nucleus egg mother cell can place suitable medium, as adds the CRlaa of 10% serum.
Among the present invention, the preferably ripe beginning back stoning in about 10-40 hour time outside the people of acceptor ovocyte more preferably began the back about 16-24 hour at maturation in vitro, and most preferably maturation in vitro began the back about 16-18 hour.
Be transferred the ovum week in the crack that enters the non-nucleus egg mother cell that is used for producing NT unit subsequently with non-nucleus egg mother cell single mammalian cell mutually of the same race.According to methods known in the art, mammalian cell and non-nucleus egg mother cell will be used to produce NT unit.For example, adopt electricity to merge fused cell.Merge by providing the electricimpulse that is enough to cause the plasma membrane instantaneous breakdown to finish electricity.Because film is repaired rapidly, plasma membrane punctures very of short duration.Therefore, if two adjacent films by induced breakdown and their lipid bilayer mix repair after, two intercellular passage aisles will be opened.Because the thermodynamic phase of these little openings, it enlarges and becomes one up to two cells.The United States Patent (USP) 4,997,384 of Prather etc. (is incorporated herein by reference at this) as a reference in full is used for further discussing this method.Available multiple electricity merge medium comprise as, sucrose, N.F,USP MANNITOL, Sorbitol Powder and phosphate buffer soln.Also can use Sendai virus and finish fusion (Graham, Wistor Inot.Symp.Monogr., 9,19,1969) as fusogen.
And (when for example using less donor nuclei) is injected directly into nuclear that merge than electricity consumption in the ovocyte may be more preferred in some cases.This technology is at Collas and Barnes, and Mol.Reprod.Dev. openly, here quotes as a reference among the 38:264-267 (1994) fully.
Preferably, mammalian cell and ovocyte oocyte maturation begin the back about 24 hours, merge medium (0.25M D-Sorbitol Powder containing, 100 μ m lime acetates, 0.5mM magnesium acetate, 1.0g/L BSA (FAF) pH7.2) μ M chambers 500 in, use electricimpulse, about 15 microseconds of 90-120V and carry out electricity fusion.After the fusion, the NT unit of the fusion that obtains just places suitable medium such as Crlaa substratum until activation.Typical activation is very short time thereafter, typically is less than 24 hours, finishes after preferably about 2～9 hours.
NT unit can activate by known method.These methods comprise, for example, cultivate NT unit at inferior physiological temp, and are cold by using in fact, or the in fact cool cold NT unit of hitting of temperature.Most convenient ground can be by reaching this purpose in incubated at room temperature NT unit, room temperature with respect to embryo the normal physiological temp condition that exposes be colder.
Optionally, can utilize known activating reagent to finish activation.For example, sperm penetrates ovocyte and has shown gestation with the multiclass gene consistent calf of pre-activated fusion ovocyte to obtain in nuclear transplantation more can survive in fertilization process.Also have, treatment process can be used to merging postactivated NT embryo as electricity and chemical shock.The activation method of suitable ovocyte is the content of the U.S. Patent number 5,496,720 of Susk-Partish etc., is incorporated herein by reference in full at this.
In addition, activation also can simultaneously or be carried out subsequently:
(i) level of divalent cation in the raising ovocyte, and
(ii) reduce the phosphorylation of cell protein in the ovocyte.This generally can lead to and divalent cation is imported ovocyte tenuigenin carries out, as, magnesium, strontium, admire or calcium, as with ionophoric form.The method of other rising divalent cation levels comprises the application electroshock, handles with Ethanol Treatment with the sequestrant of catching.
Can adopt known method to reduce phosphorylation, as, add kinase inhibitor, as serine-threonine kinase inhibitor such as 6-dimethylamino-purine, Staurosporine, 2-aminopurine and sphingosine.
Optionally, Phosphoric acid esterase and Phosphoric acid esterase 2A and Phosphoric acid esterase 2B can be imported the phosphorylation that ovocyte suppresses cell protein.
In the embodiment, can adopt after fusion in about 24 hours, and in about 2-9 hour the NT unit of merging is exposed in the TL-HEPES substratum that contains 5 μ M ionomycins and 1mg/mlBSA momently behind preferred the fusion, in the TL-HEPES that contains 30mg/ml BSA, wash the activation of finishing NT subsequently.
Activatory NT unit cultivates subsequently in suitable in-vitro culture medium and produces up to CICM cell and cell colony.Well known embryo culture and the sophisticated substratum of being suitable for.The example of the known substratum that can be used for the ox embryo culture and keep comprises that Ham ' s F-10 adds 10% foetal calf serum (FCS), tissue culture medium (TCM)-199 (TCM-199) adds 10% foetal calf serum, Di Luode-albumin-lactic acid salt-pyruvate salt (TALP), Dulbecco ' s phosphate buffered saline (PBS) (PBS), Eagle ' s and Whitten ' s substratum.Be used for one of oocytes collection and sophisticated prevailing substratum and be TCM-199 and be supplemented with 1-20% serum comprising foetal calf serum, new ox calf serum, oestrus cow serum, little sheep blood serum or bull serum.Preferably keep substratum and comprise the TCM-199 substratum that contains Earl salt, 10% bovine serum, 0.2mM Sodium.alpha.-ketopropionate and 50 μ g/ml gentamicin sulphates.Any one also can participate in the co-cultivation with various kinds of cell type such as granulosa cell, oviduct cell, BRL cell and uterine cell and STO cell in above-mentioned.
The Roenkrans that is hereby incorporated by has described another kind in the United States Patent (USP) 5,096,822 of Jr etc. and has kept substratum.The embryo culture medium of this CRl by name contains the necessary nutritive substance of embryo support.
The CRl amount is from 1.0mM to 10mM, and preferred 1.0mM is to L-lactic acid half calcium of 5.0mM.L-lactic acid half calcium is to have half calcium salt and its bonded L-lactic acid.L-lactic acid half calcium is important, mainly asks because a single composition satisfies two of substratum: (i) be used for closely cytoskeleton and arrange necessary calcium demand; The (ii) necessary lactic acid demand of metabolism and electron transport.L-lactic acid half calcium also can be used as embryo the survive mineral substance and the energy derive of necessary substratum.
Advantageously, the CRl substratum does not contain serum, as foetal calf serum, and does not need to use that zooblast is cultivated altogether or the other biological substratum, promptly contains the substratum of zooblast such as oviduct cell.Biological medium sometimes may be disadvantageous, and is deleterious and be difficult to the microorganism or the micro-factor that detect, identify and eliminate to the embryo because they may contain.
The example of the main component in the CRl substratum comprises the bovine serum albumin (sigma A-6003) of L-lactic acid half calcium, sodium-chlor, Repone K, supercarbonate and micro-FAF.Essential and the non-essential amino acid that can in substratum, add in addition, specified amount.Containing amino acid whose CRl knows with abbreviation " CRlaa ".
The CRl substratum preferably contains the following ingredients of following content:
Sodium-chlor-114.7mM
Repone K-3.1mM
Sodium bicarbonate-26.2mM
L-lactic acid half calcium-5mM
BSA-the 3mg/ml of fatty acids not
In one embodiment, activatory NT embryo unit was placed the CRlaa substratum that contains 1.9mM DMAP about 4 hours, in HECM, wash subsequently and in containing the CRlaa of BSA, cultivate then.
For example, activatory NT unit can be transferred in the CRlaa substratum that contains 2.0mM DMAP (Sigma) and at 38.5 ℃ according to appointment of envrionment conditionss, 5%CO
2Under cultivated appropriate time 4-5 hour according to appointment.
Afterwards, preferably wash the NT unit of cultivation and with being placed on during suitable medium cultivates as the CRlaa that contains 10%FCS, and 6mg/ml is included in the orifice plate that preferably contains suitable fusion feeder layer.Suitable feeder layer comprises that for example, inoblast, STO and the SI-m220 feeder cell of inoblast of the own hoof animal of penta fibrocyte Tathagata and uterine epithelial cell, chick fibroblast, mouse (as mouse or rat) are, and the BRL cell.
In one embodiment, feeder cell comprise mouse embryo fibroblasts.The embodiment that the description of suitable inoblast feeder layer preparation is seen below and in those skilled in the art, knowing.
At feeder layer (5 * 10
5Cell/ml) is gone up and is cultivated NT unit and reach the size that is suitable for transferring to recipient female up to NT unit, perhaps is suitable for obtaining can be used for producing the size of the cell of CICM cell or cell colony.Preferably, cultivate these NT cells until at least about 2-400 cell, more preferably about 4-128 cell, and most preferably at least about 50 cells.Cultivation under appropriate condition, that is, and about 38.5 ℃ and 5%CO
2Carry out, be to optimize growth, typically every approximately 2-5 days, preferred per approximately 3 days replacing substratum.
The method that is used for the processing of embryo transfer and receptor among the present invention is the used standard method of embryo transfer industry.Transplant synchronously for of the present invention successfully be important, promptly the oestrus cycle of NT embryo's period and recipient female animal is synchronous.Advantage and how supporting is estimated by uncle to see Sieolel, G.E.Jr. (" the evaluation comment of ox embryo transplantation method " (1981) L.Mastroianni in the in vitro fertilization and fetal development, Jr and J.D.Biggers compile, Plenum press, New York, NY, 323 pages), its content is incorporated herein by reference.
By the present invention, be used for having of one's own somatic karyon cloning efficiency may with use identical from the karyon of fetal cell.For example, use from cow fetus and the stacked mulberry of adult cells whose development embryonic stage identical with blastocyst stage embryo's efficient.
The present invention also can be used for cloning genetic engineering or transgenic pig.As explained above, to have advantage be because transgenic method can be simplified by the cell source of differentiation that can clonal propagation is operated in the present invention.Especially, the required dna sequence dna that has insertion, removal or modification as the noble cells of donor nuclei.Then cell those hereditary changes, differentiation is used for the nuclear transplantation of enucleation oocyte.
Any method that becomes known for inserting, remove or modify the target DNA sequence of mammalian cell all can be used for changing the noble cells of desire as nuclear donor.These methods can be removed all or part dna sequence dna, and dna sequence dna can be allogenic.Included technology has homologous recombination, one or more sequences of one or more specific site insertions, removal or modifying DNA that can be in cellular genome.
Therefore the present invention can be used for providing and has a required genotypic adult pig.The increase that has the adult pig of definite genetic dominance or other required proterties is useful especially, comprises transgenosis or genetic engineering animal, and chimaeric animals.Therefore, the present invention can produce other offspring of monotypism, also can produce the pig that meat production increases, has the proterties and the disease resistance of reproduction.And,, comprise cell of transgenosis and/or chimeric fetus and cell, tissue and the organ transplantation that tissue can be used for treating the multiple disease that is associated with use CICM cell as described below from the NT fetus.Therefore, transgenic pig has the purposes as the model of xenotransplantation that comprises disease, cell and organ and production pharmaceutical protein.
For producing CICM cell and clone, after the NT unit that obtains desirable amount, cell is removed in manual operations from the zone, uses then.This process is preferably undertaken by following steps: get the cell mass that contains NT unit, this NT unit example ground comprises at least about 50 cells, washs these cells, and with the cell plating on feeder layer, Kuo San inoblast for example.Typically, the cell that is used to obtain stem cell or cell colony can obtain from the innermost part of the NT unit that 50 cell sizes are preferably arranged of cultivating at least.But, the NT unit of less or greater number cell or also can be used for obtaining ES cell and cell colony from the cell of the other parts of NT unit.Cell is maintained suitable growth medium, for example, replenish with in the feeder layer among the α-MEM of 10%FCS and 0.1mM beta-mercaptoethanol (Sigma) and L-glutaminate.In order to help growth replaceable substratum when the needs, changed once in for example about every 2-3 days.
This cultural method causes the formation of CICM cell or clone.Those skilled in the art can change the growth that culture condition is beneficial to specific CICM cell when needing.And, can produce the CICM cell of genetic engineering or transgenic pig according to the present invention.That is, said method can be used for producing the NT unit that has introduced one or more required dna sequence dnas, perhaps produces all or part of NT unit of having removed or having modified of wherein one or more endogenous dna sequence dnas.Those genetic engineerings or transgenosis NT unit can be used for producing the cell that genetic engineering or transgenosis CICM cell comprise the people then.
The CICM cell and the cell that obtain, preferred people's CICM cell and clone have in many treatments and the purposes in the diagnosis.Its specifically, these CICM cells can be used for cellular transplantation therapy.People CICM cell can be used for the treatment of numerous disease.People NT unit itself also can be used for treatment of diseases.
Consider this point, the embryo of known mouse does (ES) cell can be divided into almost any cell type, for example hemopoietic stem cell.Therefore, the CICM cell of the pig that produces according to the present invention should have similar differentiation capability.CICM cell of the present invention can break up through inducing, to obtain required cell type according to currently known methods.For example, by these cells of cultivation in division culture medium and under the confession condition of cytodifferentiation, but the pig CICM cytodifferentiation of induction experiment becomes hemopoietic stem cell, neurocyte, muscle cell, myocardial cell, liver cell, chondrocyte, epithelial cell, urethra cell, neurocyte etc.Cause the substratum and the method for CICM cytodifferentiation known in the art, be called suitable culture condition.
For example, Palacios etc. report to have taught among the 92:7530-7537 (1995) by stem cell being carried out following inducing in institute of NAS and produce hemopoietic stem cell from embryonic lineages, this induction method comprises: at first the aggregate of these cells is cultivated in the suspension culture base that lacks vitamin A acid, and then be incubated in the same medium that contains vitamin A acid, again cell aggregation is transferred on the matrix that makes cell attachment.
And, Pedersen, J.Reprod.Fertil.Dev., 6:543-552 (1994) is the survey article with reference to a large amount of articles, and the article of institute's reference discloses the method that cell type that embryonic stem cell produces multiple differentiation in vitro differentiation comprises hemopoietic stem cell, muscle, cardiac muscle, neurocyte etc.
And Bain etc. make progress at biology, have taught among the 168:342-357 (1995) to make embryonic stem cell produce the method for the neurocyte with neuron behavior in vitro differentiation.These reference are examples of the method that obtains noble cells from embryo or stem cell reported.The particularly wherein disclosed content that relates to the method that makes the embryonic stem cell differentiation of these reference is here quoted as a reference fully.
Therefore, use known method and substratum, those skilled in the art can cultivate the CICM cell of this theme, comprise genetic engineering or genetically modified CICM cell, to obtain required differentiated cell types, and for example neurocyte, muscle cell, hematopoietic cell etc.
The CICM cell of this theme can be used for obtaining any required differentiated cell types.The therepic use of these noble cellss is unprecedented.For example, hemopoietic stem cell can be used in the therapeutic treatment that needs bone marrow transplantation.This method can be used to treat multiple disease, for example terminal cancer such as ovarian cancer and leukemia, and immune disease of mediation such as acquired immune deficiency syndrome (AIDS).Hemopoietic stem cell is obtainable, for example can be by ripe somatocyte such as epithelial cell or lymphocyte and non-nucleus egg mother cell fusion with cancer or acquired immune deficiency syndrome (AIDS) patient, obtain aforesaid CICM cell, and being beneficial to this cell of cultivation under the condition of differentiation, until obtaining hemopoietic stem cell.Such hemopoietic stem cell can be used in the treatment of diseases that comprises cancer and acquired immune deficiency syndrome (AIDS).
Optionally, can merge with non-nucleus egg mother cell, therefrom obtain people's CICM cell from the patient's of nervous disorder adult body cell, and with these cell cultures under differentiation condition to produce neuronal cell line.Comprise for example Parkinson's disease, alzheimer's disease, ALS and cerebral palsy etc. by the specified disease of transplanting these human nerve cell treatments.In Parkinsonian special case, shown that the fetal brain neurocyte of transplanting normally is connected with on every side cell and produces Dopamine HCL.This can reverse symptoms of Parkinson's disease for a long time.
Remarkable advantage of the present invention has provided the main unlimited supply of the cell that waits gene or isogenic people that is suitable for transplanting.Therefore, it will eliminate the major issue relevant with existing implantation method promptly, because the host is to graft or transplant the repulsion of the contingent transplanted tissue that the repulsion to the host causes.Traditionally, by resisting-repel medicine such as S-Neoral to prevent or reducing repulsion.Yet these medicines have significant adverse side effect such as immunosuppression, carinogenicity and very expensive.The present invention should eliminate, and perhaps significantly reduces the requirement to anti-rejection drugs at least.
Other available disease and illness of gene cell treatment of waiting comprises that for example Spinal injury, multiple sclerosis, muscular dystrophy, diabetes, hepatic diseases are hypercholesterolemia, heart disease, cartilage displacement, burn, ulcer of foot, gastrointestinal illness, vascular disease, kidney disease, urethral disease and aged relative disease and illness.
This method can be used for replacing dcc gene, as, the immunity system gene of defective, cystic fibrosis gene perhaps having imported to the gene that protein useful on making a study of subjects such as somatomedin, lymphokine, cytokine, enzyme etc. are expressed.For example, the dna sequence dna of coding brain-derived growth factor can be imported people CICM cell, these cytodifferentiation neuroblasts, and with these nerve cells transplantations to patient Parkinson to stop the forfeiture of neurocyte in the lysis.
In the past, be different with BDNF cells transfected type primary cell line with fixed cell system, not the cell with regard to right and wrong nerve (sarcoplast and inoblast) source in neural source.For example, adopted retroviral vector BDNF gene transfection astroglia cell, and these Transplanted cellss have been entered (Yoshimoto etc., brain research, 691:25-36, (1995)) in the Parkinsonian rat model.
Transplant after 32 days, the treatment of this ex vivo has reduced the parkinson symptom of rat up to 45%.Also have, Tyrosine Hydroxylase Gene is inserted astroglia cell similar result (Lundberg etc., developmental neurobiology, 139:39-53 (1996) and the reference of wherein introducing).
Yet this is from from intravital system problem being arranged.Particularly, at present the used retroviral vector gene reducing in vivo and shift be transient expression (Mulligan, summary, science, 260:26-932 (1993)).And, used primary cell in these researchs, lifetime that astroglia cell is limited and duplicating slowly.These characteristics have influenced the speed of transfection unfriendly and have hindered the screening of the cell of stable transfection.And the primary cell of breeding gene targeting used in a large amount of homologous recombination techniques almost is impossible.On the contrary, the cell of applications exploiting differentiation and the mammiferous clone of CICM cell should eliminate relevant with the retrovirus system.
The dna sequence dna that can introduce the CICM cell of this theme comprises, for example, the dna sequence dna of the enzyme of those coding schedule skin growth factors, Prostatropin, neuroglia deutero-neurotrophic somatomedin, insulinoid somatomedin (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine (interleukin, Interferon, rabbit, G CFS, tumour necrosis factor (α and β) etc.), treatment usefulness etc.
The application of people's NT unit and CICM cell, the present invention also comprises the application of cell inhuman in people's the disease treatment in cell, tissue and organ transplantation.Therefore, CICM cell, NT fetus and NT that plants arbitrarily and chimeric offspring (genetically modified or not genetically modified) can be used for the treatment that cell, tissue and organ transplantation are proper human diseases.In general, can be used between (from body, isogenic or allotransplantation) mutually of the same race or kind and the kind (xenotransplantation) according to CICM cell of the present invention, fetus and offspring.For example, the brain cell from ox NT fetus can be used for treating Parkinson's disease.
And the CICM cell of this theme also can be used as differentiation, particularly regulates the external model of the research of relevant gene with early development.And the cell, tissue and the organ that go out with the CICM cytodifferentiation of this theme also can be used in the drug research.
And the CICM cell of this theme can be used as the nuclear donor that produces other CICM cell and cell colony.
In order more clearly to describe the present invention, the following examples are provided.
Embodiment 1Ox separates with pig embryo and the fibroblastic primary culture of adult ox
The fibroblastic primary culture of ox and pig obtains from fetus (ox became pregnant 45 days and pig fetus 35 days).Aseptic removal head, liver, heart and digestive tube, chopping fetus and at the trypsinase EDTA of pre-incubation solution (0.05% trypsinase/0.02%EDTA; GIBCO, Grand Island cultivated 30 minutes in 37 ℃ in NY).Be inoculated in inoblast in the tissue culture ware and containing 10% foetal calf serum (FCS) (Hyclone, Logen, UT), α-MEM substratum (BioWhittaker, Walkersville, MD) the middle cultivation of penicillin (100IU/ml) and Streptomycin sulphate (50 μ l/ml).Inoblast is containing 5%CO under 37 ℃ of wet condition
2Air in cultivate and keep.Cell reaches that routine goes down to posterity when being paved with.
Adult fibroblast separates with skin from the lung in milk cow (about five years old age).The chopping lung tissue in 10 ℃ at trypsinase EDTA solution (0.05% trypsinase/0.02%EDTA; GIBCO, GrandIsland, NY) middle overnight incubation.Second day, the tissue and the cell of any disengaging in 37 ℃ at the trypsinase EDTA of pre-incubation solution (0.05% trypsinase/0.02%EDTA; GIBCO, GrandIsland cultivated 1 hour in NY) and carries out three continuous washing and trypsinase is cultivated (1 hour).Be inoculated in inoblast in the tissue culture ware and at the α that augments-MEM substratum (BioWhittaker, Walkersville cultivates in MDC) approximately from the blastodisc after date and grows for some time (12-15 days to 10 years old-15 years old animal of ox after fertilization) to the animal Adulthood.This method also can be used for comprising that from other Mammalss mouse is separated into fibrocyte.
Marker gene (external allogeneic dna sequence DNA) is imported embryo and adult fibroblast.
Embryo (ox and pig) and adult (ox) inoblast are carried out following electroporation operation.The standard micro-injection method also can be used for allogeneic dna sequence DNA is imported inoblast, yet, because operation is easier, so adopt the electroporation operation in the present embodiment.
To contain fibroblastic culture plate of breeding and place trypsinase EDTA solution (0.05% trypsinase/0.02/EDTA; GIBCO, Grand Island, NY) the middle cultivation becomes individual cells suspension until cell.The rotation sedimentation cell also makes every milliliter to contain 5 * 10 with phosphate buffered saline (PBS) (PBS) suspension cell again under 500xg
6Individual cell.
The reporter gene construct comprises cytomegalovirus promoter and beta-galactosidase enzymes, neomycin phosphotransferase fusion gene (β-GEO).With reporter gene and final concentration is that the cell of 50 μ g/ml is added in the electroporation cell.After the electroporation pulse, with inoblast shift get back to growth medium (contain 10% foetal calf serum (FCS) (Hyclone, Logen, UT), the α-MEM substratum (BioWhittaker of penicillin (100IU/ml), Streptomycin sulphate (50 μ l/ml), Walkersville, MFD)) in.
After the electroporation, screen adherent inoblast and be used for the stable integration of reporter gene.G418 (400 μ g/ml) is added 15 days (scopes: 3 days life end of term up to cultured cells) of effect in the growth medium.This medicine kills all cells that do not have β-GEO gene, and reason is that they are not expressed neo resistance base and exist.In latter stage during this period of time, the colony of stable transgenic cell appears.Breeding independently between each colony is mutual.With X-gal with transgenosis inoblast dyeing with the expression of observing beta-galactosidase enzymes and turn out to be the positive be used to use β-GEO gene pcr amplification integration and on sepharose, run band.
In the nuclear transplantation method, use the transgenosis inoblast to create CICM clone and transgenosis fetus
To derive from a clone (CL-1) of a colony behind the ox embryo fibroblast as the donor karyon in nuclear transplantation (NT) method.The general method of NT has been described above.
The slaughterhouse ovocyte is at maturation in vitro.The micropipette stoning that these ovocytes are peelled off upright cell and about 18 to 20 hours (hpm) uses inclination after maturation.Add Hoechst 33342 (3 μ g/ml at the TL-HEPES substratum; Sigma) confirm stoning in.Then single donorcells (inoblast) is placed the ovum week crack of acceptor ovocyte.Answer the electricity consumption integration technology that bovine oocyte tenuigenin and donor nuclei (NT unit) are merged.Being full of the once fusion pulse that acts on NT unit in the gap cell that merges substratum at 500 μ m is 120V 15 seconds.This occurs in ripe back 24hpm.Place the CRlaa substratum up to 26 to 27hpm NT unit.
The general method that is used for manually activating ovocyte has been described above.The activation of NT unit is 26 and begin during 27hpm.In brief, NT unit is exposed to ionomycin (5 μ M among the TL-HEPES that contains 1mg/ml BSA; CalBiochem, La Jolla, CA) 4 minutes and washed 5 minutes with the TL-HEPES that contains 30mg/ml BSA.In the ionomycin treating processes, also NT unit is placed 2mM DMAP (Sigma).After the flushing, NT unit is transferred in the CRlaa substratum droplet that contains 2mMDMAP (Sigma) and at 38.5 ℃, 5%CO
2Under cultivated 4 to 5 hours.Wash the embryo and place the CRlaa substratum of four well culture plates that contain mouse embryo fibroblasts fusion feeder layer to add 10%FCS and 6mg/ml BSA then.At 38.5 ℃ and 5%CO
2Cultivate NT unit under the condition more than 3 days.Changed a subculture in per three days up to activating back 5 to 8 days.This moment, protoblast phase NT embryo can be used for production transgenosis CICM (inner cell mass of cultivation) clone or fetus.The inner cell mass of separable these NT units also is planted on the feeder layer.And, NT unit is transferred in the recipient female animal.In the back interruption of pregnancy in 35-48 days of becoming pregnant.This produces the transgenosis fetus that has 7 clones of β-GEO gene in the tissue of all inspections.Detecting in 7 fetuses by ultrasonic observation has 6 to have normal heartbeat.And the tissue slice of fetus does not show significantly unusual.This method generally helps gene targeting CICM clone and fetus.
Following table has been summed up these result of experiment.
| The donorcells type | n | The spilting of an egg (%) | Protoblast (%) | CICM system * (％) | The transgenosis fetus (%) that regains | Continue gestation after 40 days |
| CL-1 ox tire inoblast (bGEO) | 412 | 220(53％) | 40(10％) | 22(55％) | N/A | N/A |
| CL-1 ox tire inoblast (bGEO) | 3625 | 2127(59％) | 46(9％) | N/A | 7 fetuses + | 8 ≠ |
| CICM clone from CL-1 NT embryo | 709 | 5(0.7％) | N/A | 0 | 0 | |
| The ox inoblast grows up | 215 | 119(55％) | 20(9％) | N/A | N/A | N/A |
* 19 systems are β-GEO positives, 2 feminine genders and a death before PCR detects.
In 35 days the growth of+gestation, a foetal death, another growth is slightly slow.5 fetuses that gestation regained in 38-45 days are normal.All fetuses turn out to be genetically modified.
≠ first offspring is born in October, 1997.
Embodiment 2Derive from the chimeric fetus and the offspring of transgenosis CICM cell.Transgenosis CICM clone is at first from transgenosis NT unit (noble cells).
The CICM clone that derives from transgenosis NT embryo (the CL-1 cell transfer is entered non-nucleus egg mother cell) is used for producing chimeric embryo and fetus.With 1-5mg/ml PRONASE A or 0.05% trypsinase/EDTA in conjunction with the depolymerization of mechanical depolymerization method render transgenic CICM cell colony, so that produce 5 or cell mass still less.Make trypsinase or the active inactivation of PRONASE A by repeatedly wash cell with 30～100% foetal calf serums.The cell of these depolymerization is placed the micrurgy plate that contains the TL-HEPES substratum.Also the embryo who is fertilized is placed these plates and uses the micrurgy instrument and produce chimeric embryo.8 to 10 transgenosis CICM injection cells are entered among the fertilization embryo of 8-16 cell stage.These embryos of vitro culture are to blastocyst stage and transplant subsequently and enter in the receptor.
6 the non-surgery of blastocyst stage chimeric embryo ground are transplanted and are entered in two recipient female animal altogether.After conceived 5 weeks, regain 3 fetuses.Several tissues of these three fetuses comprise that the screening of the sexual cell (expression germline mosaic) of sexual gland is by the Southern blot hybridization to segmental pcr amplification of beta-galactosidase enzymes and amplified production.In these three fetuses, two is from the transgenosis CICM cell contribution positive.These two fetuses all have transgenosis CICMcontribution sexual gland.
Allow 10 chimeric embryos to foot day, wherein give birth to the offspring for 7.Make the ear incisura and from each calf DNA isolation.Pcr amplification one, one of them turns out to be the chimeric offspring of transgenosis.
Transgenosis NT embryo is from transgenosis CICM clone.The rotation at first of transgenosis CICM clone
Gene NT unit (cell of differentiation)
Identical transgenosis CICM clone is used to produce the NT embryo.Except what be used as the fusion of donorcells and non-nucleus egg mother cell is CICM cell rather than the inoblast, and having used embodiment 1 is described NT method.With 1-5mg/ml PRONASE A or 0.05% trypsinase/EDTA in conjunction with the depolymerization method render transgenic CICM cell colony depolymerization of machinery so that produce 5 or still less cell mass.It enters Transplanted cells before the enucleation oocyte, and the repeatedly flushing of carrying out the 30-100% foetal calf serum by pair cell makes trypsinase or the active inactivation of PRONASE A.Reported result's (the 3rd group) in the table 1.Five blastocyst stage embryos have been produced.
Claims (21)
1. produce the method for CICM clone, comprising:
(i) under the condition that is suitable for the formation of nuclear transplantation (NT) unit, with the required mammalian cell non-serum starved, differentiation or the cell or the nucleus stoning mammal ovocyte mutually of the same race of nucleus insertion and differentiation;
(ii) activate the nuclear transplantation unit that obtains; And
(ii) cultivate the cell that obtains from the NT unit of described cultivation to obtain CICM clone.
2. the CICM clone that obtains according to the method for claim 1.
3. in the mammalian cell of described differentiation or nucleus, insert, remove according to the process of claim 1 wherein or modify target DNA, thereby cause the generation of the NT unit of hereditary change.
4. the transgenosis CICM clone that obtains according to the method for claim 3.
5. induce the CICM clone differentiation that obtains according to the process of claim 1 wherein.
6. the people's noble cells that obtains according to the method for claim 5.
7. a methods of treatment comprises that the patient who needs cellular transplantation therapy is with the gene noble cells that waits according to claim 6.
8. according to the method for claim 7, wherein carry out described cellular transplantation therapy to treat following disease or illness: Parkinson's disease, Huntington Chorea, alzheimer's disease, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic diseases, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, vascular disease, urethral disease, AIDS and cancer.
9. a methods of treatment comprises that the patient who needs cellular transplantation therapy is with people's noble cells according to claim 6.
10. according to the method for claim 9, wherein carry out described cellular transplantation therapy to treat following disease or illness: Parkinson's disease, Huntington Chorea, alzheimer's disease, ALS, spinal cord defective or damage, multiple sclerosis, muscular dystrophy, cystic fibrosis, hepatic diseases, diabetes, heart disease, cartilage defects or damage, burn, ulcer of foot, vascular disease, urethral disease, AIDS and cancer.
11. according to the method for claim 7, wherein Fen Hua people's cell is hematopoietic cell or neurocyte.
12. according to the method for claim 7, wherein therapy is to be used for the treatment of Parkinson's disease and noble cells is a neurocyte.
13. according to the method for claim 7, wherein therapy is to be used for the treatment of cancer and noble cells is a hematopoietic cell.
14., comprise that further the NT unit with the clone combines to produce mosaic with the fertilization embryo according to the method for claim 1.
15., further comprise making chimeric CICM clone develop into chimeric embryo according to the method for claim 14.
16., further comprise making chimeric embryo develop into chimeric fetus according to the method for claim 15.
17., further comprise making chimeric fetation become chimeric offspring according to the method for claim 16.
18. according to the method for claim 14, wherein in the mammalian cell of described differentiation or nucleus, insert, remove or modify target DNA, cause the generation of the NT unit of hereditary change thus.
19., further comprise making chimeric CICM cell development become chimeric embryo according to the method for claim 18.
20., further comprise making chimeric embryo develop into chimeric fetus according to the method for claim 19.
21., comprise that further chimeric fetation becomes chimeric offspring according to the method for claim 20.
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| RU2216592C2 (en) * | 1998-01-16 | 2003-11-20 | Агробиоген Гмбх | Method for obtaining animal embryos and method for raising animals out of embryos |
| US6331659B1 (en) | 1998-01-21 | 2001-12-18 | University Of Hawaii | Cumulus cells as nuclear donors |
| WO1999046982A1 (en) * | 1998-03-16 | 1999-09-23 | Relag Pty Ltd | Porcine nuclear transfer |
| AUPP294898A0 (en) * | 1998-04-15 | 1998-05-07 | Monash University | Method of nuclear transfer |
| EP1818397A1 (en) * | 1998-11-02 | 2007-08-15 | Trustees Of Tufts College | Methods for cloning animals |
| WO2000025578A2 (en) * | 1999-04-26 | 2000-05-11 | Trustees Of Tufts College | Methods for cloning animals |
| EP1375654A3 (en) * | 1998-11-02 | 2008-01-16 | GTC Biotherapeutics, Inc. | Transgenic and cloned mammals |
| US6781030B1 (en) | 1998-11-02 | 2004-08-24 | Trustee Of Tufts College, Ballou Hall | Methods for cloning mammals using telophase oocytes |
| JP2002537785A (en) | 1999-03-04 | 2002-11-12 | ピーピーエル セラピューティクス （スコットランド） リミテッド | Genetic modification of somatic cells and their use |
| NZ508739A (en) * | 1999-06-30 | 2002-11-26 | Woo Suk Hwang | Method for producing cloned tigers by employing inter-species nuclear transplantation technique |
| EP1109891A4 (en) * | 1999-06-30 | 2004-11-17 | Hwang Woo Suk | Method for producing cloned cows |
| EP1109890A4 (en) * | 1999-06-30 | 2004-12-29 | Hwang Woo Suk | Method for producing human cloned embryos by employing inter-species nuclear transplantation technique |
| NZ518191A (en) * | 1999-10-15 | 2004-01-30 | Advanced Cell Tech Inc | Methods of producing differentiated progenitor cells and lineage-defective embryonic stem cells |
| WO2001094554A1 (en) * | 2000-06-08 | 2001-12-13 | Trostner, Jens | Method for the genetic reconstruction of human organs |
| MXPA03001915A (en) | 2000-08-03 | 2004-09-10 | Therapeutic Human Polyclonals | Production of humanized antibodies in transgenic animals. |
| FR2814642B1 (en) | 2000-10-03 | 2005-07-01 | Ass Pour Le Dev De La Rech En | TRANSGENIC MOUSE FOR THE TARGETED RECOMBINATION MEDIATED BY THE MODIFIED CRE-ER |
| EP1395652A2 (en) * | 2000-11-30 | 2004-03-10 | Stemron, Inc. | Isolated homozygous stem cells differentiated cells derived therefrom and materials and methods for making and using same |
| CN100448991C (en) * | 2000-12-22 | 2009-01-07 | 麒麟医药株式会社 | Method for cloning mammals with altered donor chromatin or donor cells |
| ATE466089T1 (en) | 2000-12-22 | 2010-05-15 | Agronomique Inst Nat Rech | POSITION-INDEPENDENT AND TISSUE-SPECIFIC EXPRESSION OF A TRANSGENE IN THE MILK OF A TRANSGENIC ANIMAL |
| WO2003025159A1 (en) | 2001-08-13 | 2003-03-27 | Embrex, Inc. | Methods for injecting avian eggs |
| NZ535367A (en) * | 2002-04-01 | 2008-01-31 | Gtc Biotherapeutics Inc | A method for selecting cell lines to be used for nuclear transfer in mammalian species |
| US7612250B2 (en) | 2002-07-29 | 2009-11-03 | Trustees Of Tufts College | Nuclear transfer embryo formation method |
| CA2661848C (en) | 2006-09-01 | 2015-02-03 | Therapeutic Human Polyclonals, Inc. | Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals |
| EP2267153A1 (en) | 2009-05-26 | 2010-12-29 | Université Claude Bernard Lyon 1 | Identification of netrin-1 receptor unc5c gene mutation in solid cancers |
| US10865383B2 (en) | 2011-07-12 | 2020-12-15 | Lineage Cell Therapeutics, Inc. | Methods and formulations for orthopedic cell therapy |
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| EP0739412B1 (en) * | 1993-12-23 | 2002-02-27 | Infigen, Inc. | ungulate EMBRYONIC STEM CELLS AS NUCLEAR DONORS AND NUCLEAR TRANSFER TECHNIQUES TO PRODUCE CHIMERIC AND TRANSGENIC ANIMALS |
| GB9517779D0 (en) | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
| GB9517780D0 (en) * | 1995-08-31 | 1995-11-01 | Roslin Inst Edinburgh | Biological manipulation |
| US5945577A (en) | 1997-01-10 | 1999-08-31 | University Of Massachusetts As Represented By Its Amherst Campus | Cloning using donor nuclei from proliferating somatic cells |
| RU2216592C2 (en) * | 1998-01-16 | 2003-11-20 | Агробиоген Гмбх | Method for obtaining animal embryos and method for raising animals out of embryos |
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| EP1017422A1 (en) | 2000-07-12 |
| BR9811657A (en) | 2000-09-05 |
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| AU8154398A (en) | 1999-01-25 |
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| WO1999001163A1 (en) | 1999-01-14 |
| EP1017422A4 (en) | 2002-10-29 |
| IL133786A0 (en) | 2001-04-30 |
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